Studies of muscle fatigue have mostly been checked under isometric exercise. However EMG signals from isotonic exercise generate uncontrollable noise, so there were difficulties in gathering reliable median frequency and muscle fatigue index if frequency analysis was equally applied in isometric exercise. This study tried to compare the differences of muscle fatigue determinant variables in terms of median frequency searching methods of EMG signal, which was estimated in isotonic exercise. To accomplish this, we determined median frequency by using different FFT intervals and overlapping ration of consecutive FFT sections under the same EMG signal, and then searched for a linear regression line, and compared initial median frequency, slope, and muscle fatigue index which were variables under the linear regression line. In result of comparison, initial median frequency was more elevated as FFT exercise interval became larger. The slope of the linear regression line showed distinguishable decreasing tendency as FFT intervals were larger and overlapping sections were smaller. Significant tendency of muscle fatigue index in FFT interval was shown by high muscle fatigue index in specific FFT intervals.